The zinc-finger transcriptional repressor Gfi-1 has a critical intrinsic role in maintaining the function of hematopoietic stem cells (HSCs) and also controls a variety of their progeny at distinct, later stages of hematopoietic differentiation. HSCs lacking Gfi-1 display severely reduced activity in transplantation assays and cannot compete with wildtype HSCs in the bone marrow. In the absence of Gfi-1, HSCs exhibit strikingly increased proliferation rates and are found more frequently in active phases of the cell cycle. This suggests that Gfi-1 functions to restrict proliferation in HSCs and contrasts findings in T-lymphocytes in which it promotes proliferation. The molecular basis of Gfi-1's action at precise stages of hematopoietic differentiation is unknown, as are the molecular mechanisms modulating its function, depending on the cellular context. Clarification of the detailed mechanisms of Gfi-1 in HSCs is pivotal for developing strategies to exploit its biology in order to manipulate stem cells. The highly complex hematopoietic phenotype that follows disruption of Gfi-1 in mice is a major obstacle in our complete understanding HSCs and their more mature descendants. We have engineered a novel, conditional mouse strain that permits temporally controlled and lineage specific disruption of Gfi-1 that will enable us to dissect the roles Gfi-1 in adult HSCs and subsequently, in specific lineages. The specific aims of this proposal are: 1) to characterize the impact of induced disruption of Gfi-1 in HSCs of adult mice, 2) to identify and validate the target genes of Gfi-1 in HSCs, and 3) to elucidate the role of Gfi-1 in B-cells. Relevance to public health: Deciphering the molecular regulation of adult hematopoietic stem cells is critical for the development of urgently needed strategies to maintain, expand, and manipulate stem cells. Furthermore, it may serve as paradigm for the biology of stem cells in other tissues, leukemia, and cancer.